PROJECT ABSTRACT Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are bacterial sexually transmitted infections (STIs), and are neglected causes of adverse neonatal outcomes. According to WHO modelling estimates, CT and NG infections are the most common STIs globally. These infections are even more common among people living with HIV. The risk of vertical transmission during delivery is about 50%. HIV-infected pregnant women with CT and/or NG infections are at increased risk of MTCT of HIV. Other studies on CT and NG infections in neonates are over 20 years old and those infections are not systematically measured in sub- Saharan Africa and are largely unknown. Few countries provide routine antenatal testing. In most countries, the syndromic approach is used, which utilizes an algorithm to classify symptoms into STI syndromes and provide standardized treatment. Syndromic management lacks specificity and causes pregnant women to be unnecessarily exposed to antibiotics, and has low sensitivity, missing asymptomatic infections, putting neonates at risk. Our proposal has three specific aims. Aim 1: We will determine the burden of CT and NG infections and correlates of infection among asymptomatic pregnant women in Gaborone, Botswana by a) using diagnostic testing to estimate the prevalence of CT and NG infections at first and third trimester antenatal visits and postnatal visit and the incidence of infections between those visits; and b) assessing the correlates of infection. Aim 2: Will compare longitudinal neonatal outcomes for pregnant women tested for CT and NG infections with women who received standard antenatal care by a) estimating the frequency of vertical transmission of CT and NG infections and neonatal outcomes and the association with testing and treatment; and b) assessing independent factors that may be predictive of adverse neonatal outcomes. Aim 3: We will assess the burden of infection and markers of inflammatory response to CT and NG infection during pregnancy and associations with vertical transmission of CT and NG by a) testing immunologic factors associated with vertical transmission of CT and NG; b) determining the association between the Xpert CT/NG assay's pathogen-specific cycle threshold value (Ct) and transmission; and c) evaluating the frequency and distribution of Sample Adequacy Control (SAC) cycle threshold values (Ct) from the Xpert CT/NG assay, and evaluate any correlations with the transmission of CT/NG to neonates. We will provide evidence to help evaluate the effects of testing on vertical transmission and clinically important neonatal health outcomes, and to evaluate and understand biological correlates of transmission. This study seeks to generate evidence to inform a larger R01 grant application.